New physical concepts that overcome the diffraction limit by using the dark and bright states of the fluorescent marker emerged in the last decade [a) S. W. Hell, Science 2007, 317, 1749-1753; b). S. W. Hell, Nature Meth. 2009, 6, 24-32; c) J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, S. W. Hell, Nature Meth. 2008, 5, 943-945]. One of them is based on the reversible saturable optically linear fluorescence transitions (RESOLFT) and operates with molecular ensembles of fluorophores. For example, stimulated emission depletion (STED) microscopy—the first concept of the RESOLFT type—uses the ground (singlet) state of the fluorophore (S0) as a dark state, and the first excited state (S1) as a bright one.
In practical applications of the STED method, a focused pulse excites fluorescence in a small spot (with dimensions limited by diffraction), and immediately after that a red-shifted doughnut-shaped STED beam quenches the fluorescence of excited molecules by stimulated emission (S1→S0) everywhere, except the very centre of the doughnut. For squeezing the fluorescence to a very small central spot, the quenching rate should exceed the rate of the spontaneous transition to a ground state S0. Fluorescent lifetimes of organic fluorophores (τfl˜10−9 s) and their optical cross-sections of the S1→S0 transitions (σ˜10−16 cm2) imply that the STED-pulse should have a very high power of ca. 10 MW/cm2 [ISTED>IS≈(στfl)−1˜1025 photons/(cm2×s)]. Quite recently first STED images of the living cells were recorded at a rate of 28 frames per seconds. In the course of this study, the movements of synaptic vesicles inside the axons of cultured neurons were resolved with a precision of ca. 60 nm [V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, Science 2008, 320, 246-249].
The huge light intensities cause photobleaching of fluorophores, and therefore STED microscopy ultimately requires most photostable fluorescent dyes. For example, fluorescein derivatives are not photostable enough under severe irradiation conditions. Other important qualities of the STED dyes include high fluorescent quantum yields and oscillator strengths (high absorption coefficients), low rate of the triplet state formation, high solubility in water or aqueous buffers and a reactive group (with a linker) for attaching a dye to a biological object. Another valuable feature of a fluorescent dye is the ability to cross the cell membrane. For that, its molecule should not be too big; however a priori it is difficult to predict, if any particular dye will penetrate through the cell membrane.
If an imaging procedure provides an optical resolution on the molecular scale, or if only single molecules remain in the effective detection volume, the dye should be suitable for single molecule detection (e.g. in the method of fluorescence correlation spectroscopy—FCS).
Multicolor RESOLFT techniques and colocalization studies in biology demand improved water-soluble, exceptionally photostable and highly fluorescent dyes, which may be optically separated from each other.
Recently, a far-field fluorescence “nanoscopy” based on switching the majority of the fluorescent molecules to a metastable dark state, such as the triplet, and calculating the position of those left or those that spontaneously returned to the ground state, has been introduced (J. Fölling et. al, Nature Meth. 2008, 5, 943-945). This superresolution imaging method of the ground state depletion and single molecule return (GSDIM) requires photostable fluorescent dyes with recovery times from several tens to several hundreds of milliseconds, minimal content of the dye in the ground state after the pump pulse and the possibility to enhance the recovery by the irradiation with the UV laser (375 nm).
In the prior art, several classes of organic substances are known as fluorescent dyes: e.g. pyrenes and other condensed polycyclic aromatic compounds, rhodols, rhodamines, BODIPY derivatives, coumarines, etc.
Coumarine dyes, in particular 7-amino-4-methylcoumarines, have found a limited use as fluorescent labels, because their spectral properties cannot be easily tuned or tailor-made. Simple coumarines absorb in the near-UV region and strongly emit the violet or blue light.
Pyrenes and other condensed aromatic hydrocarbons are highly lipophilic crystalline materials, and it is difficult to increase their solubility in water in order to prevent aggregation and quenching the fluorescence in aqueous medium. However, their excitation/-emission bands can easily be shifted within the whole visible spectral range by changing the number of condensed aromatic rings.
The zwitterionic rhodols, rhodamines and carbopyronines are intrinsically rather hydrophilic. Their spectra can easily be modified by changing the substituents at the xanthene (carbopyronine) fragment and/or in the o-substituted benzoic acid residue. The same is true for the BODIPY dyes (the simplest one absorbs at about 500 nm and emits at ca. 510 nm), though they have one substantial drawback: sometimes, the fluorescence intensity of their bioconjugates is not directly proportional to a number of labelled sites [R. P. Haugland, A Guide to Fluorescent Probes and Labelling Technologies, Invitrogen Corp., Carlsbad, 2005, p. 53; for a recent review on the chemistry of fluorescent BODIPY dyes, see: G. Ulrich, R. Ziessel, A. Harrimann, Angew. Chem. Int. Ed. 2008, 47, 1184-1201]. Moreover, it is relatively difficult to chemically modify the BODIPY residue in such a way, that it becomes water-soluble. For that, many rhodols or rhodamines may easily be decorated with two sulfonic acid groups simply by sulfonation with SO3 in H2 SO4. Sulfonation is known not only to improve solubility in aqueous media, but it also considerably reduces aggregation and quenching of fluorescence in water or aqueous buffers. It often increases photostability and fluorescence quantum yield of a free dye in solution and after attaching it to a biological macromolecule.
In the following, the properties of a number of commercially available organic fluorescent dyes, in particular rhodamine dyes and fluorescein derivatives, are discussed in more detail [a) G. T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996, pp. 316-331; b) T. Karstens, K. Kobs, J. Phys. Chem. 1980, 84, 1871-1872; c) R. P. Haugland, A Guide to Fluorescent Probes and Labelling Technologies, Invitrogen Corp., Carlsbad, 2005, pp. 11-37; d) N. Panchuk-Voloshina, R. P. Haugland, J. Bishop-Stewart, M. K. Bhalgat, P. J. Millard, F. Mao, W.-Y. Leung, R. P. Haugland, J. Histochem. Cytochem. 1999, 47, 1179-1188].
One of them is rhodamine 110 (Scheme 1), which exhibits a bright green fluorescence (φfl=0.92 in basic ethanol) [R. F. Kubin, A. N. Fletcher, J. Luminescence 1982, 27, 455-462]. Positions and intensities of the absorption and fluorescent maxima of rhodamine 110 depend on the solvent and, to some extent, on pH. Structurally similar 5(6)-carboxyrhodamine 110 (known as Rhodamine Green®) possesses the second carboxylic group in the benzene ring, which may occupy either the p-position to the xanthene fragment (C-5) or to the first carboxylic group. In the latter case the second carboxylate is attached to C-6 (Scheme 1). Fluorescence of Rhodamine Green® is insensitive to pH in the range between 4 and 9. However, conjugation with proteins often quenches the fluorescence of this dye, and its conjugates may precipitate from solutions.

To make the fluorescence independent from the pH in a wider range and to reduce the fluorescence quenching after conjugation with proteins, the sulfonated version of Rhodamine Green® (3) and its amino reactive esters (4a,b) were introduced (U.S. Pat. No. 6,130,101). The second, remote carboxy group was activated in the latter compounds. It is much less sterically hindered than the carboxylate in the ortho-position to the bulky heterocyclic residue, and therefore, it may be easily and selectively activated by the formation of N-hydroxysuccinimidyl or substituted phenyl esters (4-6).

Optical properties of the commercially available derivatives of rhodamine 110 (1-6) and fluorescein (7-9, Scheme 2) are given in Table 1. Compound 5 and 6 have additional substituents in the xanthene residue—two chlorine atoms at the positions 2′ and 7′, or the condensed alkyl ring, which also incorporates one of the nitrogen atoms. These substituents shift the absorption and emission curves to the red (up to ca. 10 and 20 nm for substances 5 and 6, respectively; compared with compounds 1, 3 and 4). Interestingly, introduction of the two fluorine atoms into the positions 2′ and 7′ of fluorescein 5(6)-carboxylate (7) does not produce any spectral changes (compare the compounds 7 and 8). In order to get the bathochromic shift of ca. 10 nm (compound 9), it was necessary first to introduce 4 fluorine atoms into the o-disubstituted benzene ring of Oregon Green®, and then substitute one of them with thiol group of the mercaptoacetic acid. At the first glance, these small spectral shifts seem to be unimportant. However, there are optical devices (e.g. Zeiss META system) capable to differentiate between the fluorescence maxima, which are only 5-10 nm apart. These systems greatly enlarge the tool-box of available fluorescent colours for multicolour labelling (provided that the excitation laser is the same).
TABLE 1Optical properties of the commercially available derivatives of rhodamine110 (1-6) and fluorescein (7-9, Schemes 1 and 2).Compound1*2***3*4a*5*6*75*86*,7*96*λmax, nm4965044914945025174924*4924*506λem, nm520**5325155205255425174*5184*526ε × 10−4 8.37.87.27.18.0 7.84* 8.54*8.6Notes:*water, pH = 7;**524 nm in basic EtOH, Φfl = 0.92;***MeOH;4*H2O, pH = 9;5*fluorescence is quenched at pH < 7;6*spectra are pH-dependent at pH < 5;7*τ = 4.1 ns (20° C.).
Derivatives of rhodamine 110 (2-6) are much better fluorophores than spectrally similar fluoresceins (7-9). The parent dye—5(6)-carboxyfluorescein (7)—has relatively high rate of photobleaching, compared with Alexa Fluor® 488. Its fluorescence is highly pH-dependent and significantly reduced at pH<7. High degree of conjugation with biopolymers may quench the fluorescence, so that it will not be directly proportional to the number of fluorescein residues per protein molecule. These drawbacks are no more present in Oregon Green® 488 and Oregon Green® 514, the derivatives of fluorescein with one common feature—two fluorine atoms in the positions 2′ and 7′ of the xanthene ring (compounds 8 and 9). They are more photostable than fluorescein 7, but still bleach faster than Alexa Fluor® 488 derivatives. Moreover, higher degree of labelling proportionally increases the fluorescence of conjugates, especially in the case of Oregon Green® 514. The latter dye has approximately the same photostability as Rhodamine Green® (2).
However, all of these fluorescent dyes of the prior art have still some drawbacks with respect to photostability and other properties required for application in the most recent microscopic and spectroscopic techniques such as STED, GSDIM etc, where very high light intensities are used, as outlined above.
Consequently, the main object of the present invention was to provide novel fluorescent photostable dyes which would exhibit improved properties, namely in particular resistance against phobleaching in the presence of air-oxygen, hydrophilicity, high values for adsorption and emission maxima, recovery times of several tens—several hundreds of milliseconds, low content of the dye in the ground state after the pump pulse, the possibility of the enhanced recovery caused by the irradiation with the UV light, and which would be particularly suitable for microscopy applications with very high light intensities such as STED, FCS and GSDIM.
This object has been achieved by providing the novel fluorinated rhodamines according to claims 1-3, the methods according to claims 4-6 and the uses of claims 7-13.